Rapid, multisample isoelectric focusing in sucrose density gradients using conventional polyacrylamide electrophoresis equipment: A two-peak transient in the approach-to-equilibrium

Behnke, James; Dagher, Shawky M.; Massey, Thomas; Deal, William C.

doi:10.1016/0003-2697(75)90558-8

Cited by 49 publications

(9 citation statements)

References 22 publications

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“…The double-peak approach to equilibrium is theoretically predicted for the case of a stepless pH gradient [12] and has been experimentally verified in many configurations with carrier ampholytes [5,[10][11][12]. It is well known that the commercial carrier ampholyte mixtures comprise more than 20 components per pH unit [2].…”

Section: Comparison With Experimental Resultsmentioning

confidence: 99%

“…In an attempt to explore and describe the kinetic aspects of IEF in the presence of carrier ampholytes, Catsimpoolas developed scanning gel IEF [8] and scanning sucrose density IEF [9], techniques that led to the description of transient state IEF [10]. The thereby documented double peak approach to protein equilibrium was also studied by Behnke et al [11] in density-stabilized gradients and described theoretically by Dishon and Weiss [12]. Furthermore, Thormann et al [13][14][15] investigated protein focusing in capillaries with rectangular cross sections along which the electric field distribution could be monitored and Mosher et al [15,16] described the behavior of proteins in continuous-flow IEF.…”

Section: Introductionmentioning

confidence: 97%

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High‐resolution computer simulation of the dynamics of isoelectric focusing of proteins

et al. 2004

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A dynamic electrophoresis simulator that accepts 150 components and voltage gradients employed in the laboratory was used to provide a detailed description of the focusing process of proteins under conditions that were hitherto inaccessible. High-resolution focusing data of four hemoglobin variants in a convection-free medium are presented for pH 3-10 and pH 5-8 gradients formed with 20 and 40 carrier ampholytes/pH unit, respectively. With 300 V/cm, focusing is shown to occur within 5-10 min, whereas at 600 V/cm separation is predicted to be complete between 2.5 and 5 min. The time interval required for focusing of proteins is demonstrated to be dependent on the input protein charge data and, however less, on the properties of the carrier ampholytes. The simulation data reveal that the number of transient protein boundaries migrating from the two ends of the column towards the focusing positions is equal to the number of sample components. Each protein is being focused via the well-known double-peak approach to equilibrium, a process that is also characteristic for focusing of the carrier ampholytes. The predicted focusing dynamics for the hemoglobin variants in pH 3-10 and pH 5-8 gradients are shown to qualitatively agree well with experimental data obtained by whole-column optical imaging.

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Section: Comparison With Experimental Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

High‐resolution computer simulation of the dynamics of isoelectric focusing of proteins

et al. 2004

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“…It is unlikely that one of the minor peaks is due to a transition in the approach to equilibrium as noted by focusing experi ments in sucrose [17] or polyacrylamide gels [18], since prolonged focusing (72 h) did not change the pattern.…”

Section: Discussionmentioning

confidence: 99%

Studies on the Structure of the Two Infectious Types of Densonucleosis Virus

Tijssen

Kurstak

1979

Intervirology

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No electrophoretic differences were detected between the nucleic acids of the two types of densonucleosis virus, DNV-I and DNV-II. Both virus types had similar isoelectric points. Although the viral proteins found in each type were identical, their stoichiometry differed. The localization of the structural protein, by labeling with periodate-oxidized glycoprotein, supported the hypothesis that 60 molecules of p49 aggregated into a dodecahedron (12 pentamers), whereas the two other proteins (p59 and p69) might have a stabilizing function and were localized on the outer surface of the p49 dodecahedron.

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“…The advantages of a density gradient for high molecular weight samples are rapid recovery of samples and short focusing time. Several IEF columns of low volume have been designed for a variety of purposes (2)(3)(4)(5)(6). The low-volume density gradient column used in this study is similar to that described by Sherbet (7) for studies on animal cell surface charge.…”

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confidence: 99%

Isoelectric focusing of plant cell membranes

Griffing

Quatrano²

1984

Proc. Natl. Acad. Sci. U.S.A.

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An isoelectric focusing method is described that discriminates plant cell organelle populations on the basis of surface charge. The isoelectric points (pI values) of the Golgi apparatus, the mitochondria, and putative plasma membrane from etiolated pea stem cells are reported. -6). The low-volume density gradient column used in this study is similar to that described by Sherbet (7) for studies on animal cell surface charge. The operational characteristics are similar to the IEF-M3 column he described (7). The pI of most plant and animal organelles and membrane fractions is not known. The phase partition method of pI determination has been used to study the surface charge on the membranes of the chloroplast (8). A combination of phasepartition cross-point determination and IEF in a sucrose gradient has been used to establish the pI of animal cell mitochondria (9).Variation in membrane composition make it likely that the surface differences of organelles will be reflected as a difference in their pI values. In our studies on the polarity of zygotes of the brown alga Fucus, we attempted to apply IEF as a method to demonstrate a difference in the pI of vesicle fractions subject to polar or nonpolar transport. We report here details of this procedure and the results from the Fucus experiment. In addition, we demonstrate how the technique is applied to the determination of the pI of organelles and membrane fractions as well as its potential use for preparative separation and isolation of subcellular particles and whole plant protoplasts. MATERIALS AND METHODSPlants and Chemicals. Pea seeds (Pisum sativum L. var. Alaska) were from Burpee Seed (Riverside, CA). Pea seeds were sown in vermiculite and watered every other day with tap water. They were grown in the dark at room temperature and all manipulations were done under dim red light. After 8 days, the epicotyls were harvested and an 8-mm segment was cut from 3 mm below the apical hook after they had been abraided with emery (aluminum oxide 400, Edmund Scientific, Barrington, NJ). Fucus distichus L. receptacles were collected from Yaquina Head (Newport, OR), and treated as described for the release and synchronous development of zygotes (10). Catharanthus roseus plants were made available by F. Constabel (Plant Biotechnology Institute, National Research Council, Saskatoon, Sk., Canada). Cytochrome c (type III), dithioerythritol, UDPglucose and cellobiose were from Sigma. Biolyte carrier ampholytes 3/10, 3/5, 4/6, and 7/9 were from Bio-Rad. [1-3H]Glucose (specific activity, 4.5 Ci/mmol; 1 Ci = 37 GBq) and [14C]UDPglucose (specific activity, 275 mCi/mmol) were from ICN; H235504 (specific activity, 150 mCi/mmol) was from New England Nuclear.Mira-cloth was from Calbiochem-Behring. Glass double-distilled water was used in all gradients, assays, and homogenization media.IEF Apparatus. The apparatus shown in Fig. 1 was an adaptation of the design by Sherbet for IEF-M3 (7). In contrast to the solid-piece construction of Sherbet, two separate tubes of Plexiglas were u...

show abstract

Rapid, multisample isoelectric focusing in sucrose density gradients using conventional polyacrylamide electrophoresis equipment: A two-peak transient in the approach-to-equilibrium

Cited by 49 publications

References 22 publications

High‐resolution computer simulation of the dynamics of isoelectric focusing of proteins

High‐resolution computer simulation of the dynamics of isoelectric focusing of proteins

Studies on the Structure of the Two Infectious Types of Densonucleosis Virus

Isoelectric focusing of plant cell membranes

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